CN102676701B

CN102676701B - Universal RT-PCR (Reverse Transcription Polymerase Chain Reaction) detection primer and detection method for avian pneumovirus

Info

Publication number: CN102676701B
Application number: CN 201210190445
Authority: CN
Inventors: 孙石开; 宋永峰; 鲁俊鹏; 刘闯; 覃健萍; 陈�峰; 张祥斌; 邝春明
Original assignee: Guangdong Wens Foodstuff Group Co Ltd
Current assignee: Winson food group Limited by Share Ltd
Priority date: 2012-06-11
Filing date: 2012-06-11
Publication date: 2013-08-14
Anticipated expiration: 2032-06-11
Also published as: CN102676701A

Abstract

The invention discloses a universal RT-PCR (Reverse Transcription Polymerase Chain Reaction) detection primer and a detection method for avian pneumovirus (APV), belonging to the technical field of detection for avian viruses.. The universal RT-PCR detection primer has nucleotide sequences shown as SEQ ID NO:1 to SEQ ID NO:2. The detection method is as follows: the universal RT-PCR detection primer for avian pneumovirus and the RNA (Ribonucleic Acid) of a sample to be detected are subjected to RT-PCR, and the presence of avian pneumovirus in the sample to be detected is confirmed if the PCR product fragment is 319bp o 320bp. The method disclosed by the invention is high in primer specificity, small in fragment and high in sensitivity, can finish the entire PCR process within 2 hours and can also specifically detect the presence of various subtype APVs, thus greatly reducing the erroneous diagnosis for APV infection.

Description

The universal RT-PCR of avian pneumovirus detects primer and detection method

Technical field

The present invention relates to bird virus detection techniques field, be specifically related to the universal RT-PCR of a pair of avian pneumovirus and detect primer and detection method.

Background technology

Avian pneumovirus (Avian pneumovirus, APV) have another name called Turkey Rhinotracheitis Virus (Turkey Rhinot racheitis Virus, TRTV), belong to Paramyxoviridae Pneumovirinae Pneumovirus, for the negative burst RNA viruses of strand, can cause turkey and some other bird upper respiratory diseases.The APV genome has comprised 8 main structural protein genes, and the genome total length is about 13.4 kb, and 8 genes are held the 5' end to put in order from 3' to be N-P-M-F-M2-SH-G-L.It is believed that at first APV has only a serotype, two gene hypotypes (A and B) can effectively distinguish two genotype by nucleotide sequence analysis and monoclonal antibody analysis.Occurred two kinds of different novel APV strains (C type and D type) in the U.S. with France the end of the nineties in last century respectively, the view before having changed, and people think that thus there is the strain of four above genotype or a plurality of serotypes at least in APV.RT-PCR has been widely used in the cause of disease detection of APV, and the RT-PCR of design distinguished different subtype APV is much simpler than the universal RT-PCR of design.But hypospecificity RT-PCR can be caused usually being left in the basket when new A PV exists as the detection technique of first-selection, be unfavorable for that infection detects effectively to APV, D type APV just is realized after France exists above 15 years.Existingly detect primer for APV and can only detect wherein a certain hypotype, should virus for the ease of complete detection, be badly in need of a kind of universal RT-PCR detection method.

Summary of the invention

The objective of the invention is at above-mentioned deficiency of the prior art, provide the universal RT-PCR of a kind of avian pneumovirus to detect primer.

Another object of the present invention provides a kind of avian pneumovirus RT-PCR detection method.

The present invention is achieved through the following technical solutions above-mentioned purpose:

The universal RT-PCR of avian pneumovirus detects primer, and its nucleotide sequence is shown in SEQ ID NO:1 ~ 2.

Primer based on above-mentioned design; the present invention also protects a kind of avian pneumovirus detection method; be to carry out RT-PCR with primer shown in above-mentioned SEQ ID NO:1 ~ 2 and sample RNA to be checked, if PCR product clip size is 319bp or 320bp, then in the sample to be checked avian pneumovirus arranged.Can use 1% agarose gel electrophoresis to identify the PCR product, if electrophoresis result amplified band occurs at 319bp or 320bp place, namely showing in the sample to be checked has avian pneumovirus.

In the above-mentioned avian pneumovirus detection method, the reaction system of RT-PCR contains primer, sample RNA shown in single stage method PCR mixed enzyme (PrimeScipt 1 Step Enzyme Mix), damping fluid (2 * 1 Step Buffer), SEQ ID NO:1 ~ 2 and does not have water (the RNase Free H of RNA enzyme ₂O).

In the above-mentioned avian pneumovirus detection method, the response procedures of RT-PCR is:

（1）50℃ 30min；

(2) pre-sex change: 95 ℃ of 4min;

(3) sex change: 94 ℃ of 30sec; Annealing: 55 ~ 58 ℃ of 20sec; Extend: 72 ℃ of 1 min; Carry out 30 circulations altogether;

(4) extend: 72 ℃ of 10min.

Annealing temperature the best is 57 ℃ in the step of above-mentioned RT-PCR response procedures (3).

When above-mentioned avian pneumovirus detection method, RT-PCR amplified production sequence were in SEQ ID NO:3 ~ 6 any one, illustrating in the sample to be checked had avian pneumovirus.This detection method can detect the multiple hypotype of APV virus simultaneously, comprises A, B, four present known hypotypes of C, D at least.

Compared with prior art, the present invention has following beneficial effect:

Primer specificity height of the present invention and fragment is little, susceptibility is high, whole PCR process can be finished in 2h, can detect the existence of each hypotype APV specifically, can reduce greatly APV is infected the mistakes and omissions diagnosis.

Description of drawings

Fig. 1. each subtype virus of APV detects electrophoresis result, wherein M:DNA Marker DL2000; 1, blank; 2, A type APV; 3, Type B APV; 4, C type APV.

Fig. 2. each subtype virus RT-PCR product sequencing result of APV, wherein TN-A is the A hypotype, and TN-B is the B hypotype, and TN-C is the C hypotype, and TN-D is the D hypotype.

Embodiment

Embodiment 1

1, test materials

1.1 main agents and instrument

AxyPrep body fluid viral DNA/RNA small volume of reagent box (Cat No.:AP-MN-BF-VNA-250), TaKaRa PrimeScript 0ne step RT-PCR Kit Ver.2 (TaKaRa Code:DRR055A), the Thermo high speed low temperature centrifugal machine, Thermo PCR instrument.

1.2 strain

A, Type B APV low virulent strain are available from the biological company limited of Hai Bolai, and C type APV low virulent strain is available from the biological company limited of Cimmeria, as sample to be checked.

1.3 design of primers

At first contrast common conservative region on APV A, B, C, the D genome N gene order, the primer TN01 that to design a pair of amplification purpose band then be 319bp or 320 bp.

Table 1

2, test method

2.1 RNA template preparation

Use AxyPrep body fluid viral DNA/RNA small volume of reagent box (Cat No.:AP-MN-BF-VNA-250) to extract template ribonucleic acid (RNA of A, B, C type APV low virulent strain) ,-70 ℃ of preservations.

2.2 PCR reaction

Reaction system: use TaKaRa PrimeScript 0ne step RT-PCR Kit Ver.2 (TaKaRa Code:DRR055A) PCR test kit, reaction system is 25 μ L systems, and is specific as follows:

Sequence number	Component	Volume
				1	PrimeScipt 1 Step Enzyme Mix	1μL
2	2×1 Step Buffer	12.5μL
			3	TN01-F(10μM)	0.5μL
4	TN01-R(10μM)	0.5μL
			5	Template ribonucleic acid	5 μ L (about 500ng)
6	RNase Free H2O	Complement to 25 μ L

Reaction conditions: reverse transcription: 50 ℃ of 30min; Pre-sex change: 95 ℃ of 4min; 30 circulations of increasing (comprising: sex change: 94 ℃ of 30sec; Annealing: 57 ℃ of 20sec; Extend: 72 ℃ of 1 min); Extend at last: 72 ℃ of 10min.

The PCR product is identified through 1% agarose gel electrophoresis.

3, experimental result

Identify that through agarose gel electrophoresis the purpose band that the PCR reaction obtains meets expection size (Fig. 1), each hypotype APV strain can both amplify size and be the purpose band of 319bp or 320 bp.Show (seeing SEQ ID NO:3 ~ 5) through sequencing result, the fragment of A, B, the amplification of C hypotype is correct, and by sequence contrast (see figure 2), TN01 can also detect D type APV(in theory and see SEQ ID NO:6).

SEQUENCE LISTING

＜110〉Guangdong Wen's Food Group Co., Ltd.

＜120〉the universal RT-PCR of avian pneumovirus detects primer and detection method

<130>

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

＜213〉artificial sequence

<400> 1

gcaagcttat ggtgcaggtc 20

<210> 2

<211> 19

<212> DNA

＜213〉artificial sequence

<400> 2

tgcatagctt tctgctgca 19

<210> 3

<211> 319

<212> DNA

<213> APV-A

<400> 3

gcaagcttat ggggcagggc aaacaatgct gcgctggggt gtcattgcac gatcctccaa 60

caatataatg ttgggccatg tatctgtcca agctgagttg aggcaagtat ctgaggtcta 120

tgacctagtg aggaaaatgg gacctgagtc agggttacta cacttacgcc agagtcccaa 180

agcgggtctt ttatcattga ccaactgtcc caattttgcc agtgttgtcc tcgggaacgc 240

cgccgggctt ggtattatag gcatgtacaa aggtcgagcc cccaaccttg agctgtttgc 300

tgctgctgaa agctatgca 319

<210> 4

<211> 319

<212> DNA

<213> APV-B

<400> 4

gcaagcttat ggtgcaggtc agactatgct aagatggggt gttgtggcaa gatcatccaa 60

taacatcatg ttgggccatg tgtctgtgca ggcagagtta aggcaggtct cagaagtgta 120

tgatcttgtt aggaaaatgg gtcctgaatc aggcctcctc cacttgaggc aaagtccaaa 180

agcaggctta ctatcattaa caagttgccc caactttgca agtgttgttt tggggaatgc 240

agctggccta ggcatcattg ggatgtataa gggcagagca cccaacttgg agttgttttc 300

tgcagcagaa agctatgca 319

<210> 5

<211> 320

<212> DNA

<213> APV-C

<400> 5

gcaagcttaa tggtgcaggt caaacaatgc taaggtgggg agtgatcgca agatcttcca 60

acaatataat gttgggccat gtctctgtac aagcagaact caaacaggtc acggaggtat 120

atgatctagt tagagaaatg ggccctgagt caggtcttct ccacctgagg caaagcccca 180

aggctgggtt gttatcactt gccaattgtc caaattttgc aagtgttgtg ctagggaatg 240

cctcaggatt ggggatactt ggtatgtata gaggaagggt accaaacaca gagctgtttg 300

ctgcagcaga aagctatgca 320

<210> 6

<211> 319

<212> DNA

<213> APV-D

<400> 6

gcaggcttat ggggccggtc aaactatgct gcgatggggg gtagtagcaa ggtcctcgaa 60

taacataatg ttgggccatg tgtctgtcca agctgaactt aaacaagtgt cagaagttta 120

tgatctagtg aggaaaatgg gacccgagtc agggttgctc catctcagac aaagcccaaa 180

agcagggctt ctgtctttaa ctagctgccc caattttgct agtgtagtgc tgggtaatgc 240

tgcaggcctg ggaataatag ggatgtataa aggacgagcc cccaaccttg agctgtttgc 300

tgctgctgaa agctatgca 319

Claims

1. the universal RT-PCR of avian pneumovirus detects primer, it is characterized in that nucleotide sequence is shown in SEQ ID NO:1 ~ 2.